Brachiaria Endophytes and Related Metods

ABSTRACT

The present invention relates to endophytes, in particular endophytes associated with plants of the Brachiaria-Urochloa species complex, plants infected with the endophytes, products produced by the endophytes, and related methods.

FIELD OF THE INVENTION

The present invention relates to endophytes, plants infected with endophytes, products produced by the endophytes and related methods.

BACKGROUND OF THE INVENTION

Microbes represent an invaluable source of novel genes and compounds that have the potential to be utilised in a range of industrial sectors. Scientific literature gives numerous accounts of microbes being the primary source of antibiotics, immune-suppressants, anticancer agents and cholesterol-lowering drugs, in addition to their use in environmental decontamination and in the production of food and cosmetics.

A relatively unexplored group of microbes known as endophytes, which reside in the tissues of living plants, offer a particularly diverse source of novel compounds and genes that may provide important benefits to society, and in particular, agriculture.

Endophytes often form mutualistic relationships with their hosts, with the endophyte conferring increased fitness to the host, often through the production of defense compounds. At the same time, the host plant offers the benefits of a protected environment and nutriment to the endophyte.

Other microbes, such as bacteria which can reside in the tissues of living plants, are also relatively unexplored in this setting. Plant-borne bacteria offer similar benefits.

The Brachiaria-Urochloa species complex is a component of the grass family Poaceae, with representatives distributed throughout tropical regions, particularly in Africa. Genetic diversity analysis based on internal transcribed spacer (ITS) nuclear ribosomal DNA sequence data indicates a strong affinity between Urochloa and Brachiaria, supporting morphological and anatomical studies that show a continuous gradation between these grass genera. Some Brachiaria-Urochloa species are economically significant tropical forage grasses that have been released as commercial cultivars and include B. brizantha, B. decumbens, B. humidicola, and B. ruziziensis, as well as corresponding interspecific and intraspecific hybrids.

Methods for the identification and characterization of novel endophytes and their deployment in Brachiaria-Urochloa plant improvement programs have been discussed in WO2012/174585, the disclosure of which is hereby incorporated herein in its entirety. Strains of endophytic fungi were isolated from Brachiaria-Urochloa species. These Brachiaria fungal endophytes were genetically diverse. Some of these endophytes exhibited broad spectrum anti-fungal activity and may play a role in protecting Brachiaria-Urochloa from fungal pathogens, such as Drechslera spp., which cause leaf spots.

There remains a general lack of information and knowledge of the fungal endophytes of the Brachiaria-Urochloa species complex as well as of methods for the identification and characterisation of novel endophytes and their deployment in Brachiaria-Urochloa plant improvement programs.

There is also a general lack of information and knowledge of the bacterial endophytes of the Brachiaria-Urochloa species complex as well as of methods for the identification and characterisation of novel bacterial organisms and their deployment in Brachiaria-Urochloa plant improvement programs.

Furthermore, although widely used for pasture-based agriculture in tropical regions of South America, Asia and Australia, Brachiaria-Urochloa exhibits a number of shortcomings that constrain both its use and genetic enhancement.

Forage grasses, including Brachiaria-Urochloa, have also been recognised in recent years for implications in nitrogen pollution. A major concern of modern production in agriculture is the high level of nitrogen (N) pollution and low efficiency of N utilisation. N losses from denitrification results in environmental pollution and inefficient use of both soil N and applied N (as fertiliser).

Nitrification is carried out primarily by two groups of chemo-lithotrophic bacteria (Nitrosomonas sp. and Nitrobacter spp), ubiquitous components of the soil microbial population. For example, nitrifying soil bacteria, such as Nitrosomonas spp convert ammonium (NH₄ ⁺) to nitrate (NO₃ ⁻). Nitrate can also be converted to nitrous oxide (N₂O) gas. Inhibition of nitrification may keep N in soil for longer and improve nitrogen use efficiency (NUE).

A bioluminescence assay using a recombinant strain of Nitrosomonas europaea has been developed to detect nitrification inhibitors released from plant roots, making it possible to determine and compare the biological nitrification inhibition (BNI) capacity of different crops and pastures (Subbarao et al., 2006).

The concept of plants releasing inhibitory compounds that suppress soil nitrification has previously been suggested. Several researchers have observed a slow rate of nitrification in soils of certain tropical grassland and forest soils. BNI is the ability of certain plant species to release organic compounds from their roots that have a targeted suppressive effect on soil nitrifying bacteria (Subbarao et al., 2006 2009).

Brachialactone is the major nitrification inhibitor released from roots of B. humidicola (Subbarao et al. 2009). Brachialactone belongs to a group of diterpenes called Fusicoccanes. Fusicoccanes have been identified and isolated from a diverse range of plants, fungi and bacteria. Brachialactone has been shown to exhibit biocidal activity against Nitrosomonas spp (Subbarao et al. 2009). N is then available to plant, increasing pasture performance. Literature has suggested that this compound is produced by the plant in response to ammonium in the root environment (Subbarao et al. 2009).

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for isolating, selecting and/or characterising an endophyte strain, said method including:

-   -   providing samples of plant material from plant species of the         Brachiaria-Urochloa species complex;     -   subjecting said samples to metagenomic analysis;     -   identifying bacterial and/or fungal operational taxonomic units         (OTUs) in each Brachiaria-Urochloa plant species;     -   comparing the OTUs present in each sample species to identify         core, supplemental and/or unique microbiomes; and     -   selecting endophyte strains representing a desired core,         supplemental or unique microbiome.

By an endophyte strain is meant a bacterial or fungal strain that is closely associated with a plant, preferably a plant of the Brachiaria-Urochloa species complex. By ‘associated with’ in this context is meant that the bacteria or fungus lives on, in or in close proximity to the plant. For example, it may be endophytic, for example living within the internal tissues of the plant, or epiphytic, for example growing externally on the plant.

The plant material used to prepare the samples is from a plant of the Brachiaria-Urochloa species complex. More particularly, the plant of the Brachiaria-Urochloa species complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, Brachiaria marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa eminii, Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota, Urochloa xantholeuca, Brachiaria holosericea, Brachiaria reptans, Brachiaria milliformis, and Brachiaria distachya, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.

In a particularly preferred embodiment, the plant of the Brachiaria-Urochloa complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria ruziziensis and Urochloa mosambicensis.

Preferably, the samples of plant material are selected from the group consisting of leaf, stem, root and seed material. Even more preferably, samples of leaf, stem and root types are provided. Alternatively, samples of seed are provided. In another preferred embodiment samples of leaf, stem, root and seed material are provided.

By ‘subjecting said samples to metagenomic analysis’ as used herein is meant that metagenomic sequence data is generated from the plant material. More particularly, genetic material recovered from the plant samples is analysed to produce bacterial and/or fungal sequence data.

The term ‘recovering genetic material’ includes the extraction of genetic material, including DNA, from the sample of plant material.

The genetic material recovered from the plant samples may be enriched for DNA from endophytic strains (such as bacterial and/or fungal DNA) closely associated with the plant, as part of the process of recovering the genetic material from the sample of plant material.

Accordingly, in a preferred embodiment, the step of providing samples of plant material from plant species of the Brachiaria-Urochloa species complex includes the steps of:

-   -   grinding the plant material;     -   washing the ground plant material with alcohol; and     -   extracting nucleic acid from the alcohol wash.

In this aspect of the invention, preferably the plant material is plant seed. Preferably the plant material is roughly ground. Preferably the alcohol is ethanol, more preferably 100% ethanol. Preferably the ground plant material is washed multiple times, for example two times with alcohol. Preferably the nucleic acid is DNA.

Applicants have surprisingly found that it is possible to reduce the amount of plant nucleic acid, e.g. plant seed nucleic acid and/or enrich for nucleic acid from endophytic strains by roughly grinding the plant tissue, e.g. seed, and then washing this in alcohol, preferably ethanol. While applicants do not wish to be restricted by theory, it is thought that the alcohol acts to preserve the microbe component of the plant material, particularly seed. Extracting nucleic acid, e.g. DNA from the alcohol, e.g. ethanol, wash reduces the amount of host plant nucleic acid and enriches for microbe nucleic acid. This overcomes or at least alleviates the problem of one or more prior art methods, including those which generate large numbers of sequence reads to capture the microbial component or use differences in nucleic acid, e.g. DNA methylation density to distinguish between host and microbial nucleic acid.

In a preferred embodiment, universal polymerase chain reaction (PCR) primers for profiling bacterial microbiome and/or fungal microbiome may be used to generate the sequence data. For example, primers directed to the 16S rDNA gene, more particularly the V4 region of the 16S rDNA gene, may be used for profiling bacterial microbiome. For example, primers directed to the internal transcribed spacer (ITS) region of rDNA genes, more particularly the ITS2 region of rDNA genes, may be used for profiling fungal microbiome.

In one embodiment, bacterial sequence data is produced using universal primers directed to the V4 region of the 16S rDNA gene and the fungal sequence data is produced using universal primers directed to the ITS2 region of the rDNA genes.

Metagenomic sequence data may be assembled to create the bacterial and/or fungal operational taxonomic units (OTUs). Preferably, the metagenomic sequence data may be quality trimmed and then paired using a paired-end assembler for sequences, such as PANDAseq, to create the bacterial and/or fungal OTUs.

The OTUs may be aligned against a bacterial database, such as the GreenGenes bacterial database, and/or a fungal database, such as the UNITE fungal database, to assign taxonomy.

The number of sequences associated with OTUs may be calculated for each sample.

By comparing the OTUs present in each sample species, core, supplemental and unique microbiomes may be identified. By a ‘microbiome’ is meant the collective genomes of the bacteria and/or fungi. By a ‘core microbiome’ is meant OTUs that are found across all or substantially all Brachiaria-Urochloa species tested. By a ‘supplemental microbiome’ is meant OTUs that are found across a subset of the Brachiaria-Urochloa species tested. By a ‘unique microbiome’ is meant OTUs associated with specific Brachiaria-Urochloa species.

Endophytes having a desired core, supplemental or unique microbiome may then be selected. For example, endophytes with a broad host range may be selected as candidates for delivery of traits into plants of the Brachiaria-Urochloa species complex. For example, endophytes with a narrow or specific host range may be selected as candidates for specific traits of interest, for example for production of compounds that provide beneficial properties such as improved tolerance to water and/or nutrient stress, or improved resistance to pests and/or diseases in the plant with which the endophyte is associated. In a preferred embodiment, the beneficial properties include insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity. In a particularly preferred embodiment the compound may be an inhibitory compound, such as a nitrification inhibitor, for example a fusicoccane such as brachialactone.

In a second aspect of the present invention there is provided a substantially purified or isolated endophyte selected from the group consisting of Hypocrea sp./Acremonium sp., Acremonium sp., Microsphaeropsis arundis, and Sarocladium sp./Acremonium sp., as described herein. Preferably said endophyte is isolated, selected and/or characterised by a method as hereinbefore described.

Representative samples, namely Hypocrea sp./Acremonium sp. 2.15.A.2, Acremonium sp. 2.3.C.1, Microsphaeropsis arundis 2.10.D.1, Sarocladium sp./Acremonium sp. 2.12.B.1, Sarocladium sp./Acremonium sp. 2.10.C.2 and Sarocladium sp./Acremonium sp. 2.11.B.1 were deposited at The National. Measurement Institute on 22 Sep. 2015 with accession numbers V15/028236, V15/028237, V15/028238, V15/028239, V15/028240, V15/028241 and V15/028242, respectively.

By ‘substantially purified’ is meant that the endophyte is free of other organisms. The term therefore includes, for example, an endophyte in axenic culture. Preferably, the endophyte is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure, even more preferably at least approximately 99% pure. Preferably the endophyte is in axenic culture.

The term ‘isolated’ means that the endophyte is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring endophyte present in a living plant is not isolated, but the same endophyte separated from some or all of the coexisting materials in the natural system, is isolated.

In its natural environment, the endophyte may live mutualistically within a plant. Alternatively, the endophyte may be an epiphyte, i.e. grow attached to or upon a plant. The endophyte may be a fungal endophyte or a bacterial endophyte.

The endophyte of the present invention may, in its natural environment, be associated with a plant of the Brachiaria-Urochloa species complex. More particularly, the plant of the Brachiaria-Urochloa species complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, Brachiaria marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa eminii, Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota, Urochloa xantholeuca, Brachiaria holosericea, Brachiaria reptans, Brachiaria milliformis, and Brachiaria distachya, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.

In a particularly preferred embodiment, the plant of the Brachiaria-Urochloa complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria ruziziensis and Urochloa mosambicensis.

By ‘associated with’ in this context is meant that the endophyte lives on, in or in close proximity to the plant. For example, it may be endophytic, for example living within the internal tissues of the plant, or epiphytic, for example growing externally on the plant.

The fungus may be a heterotroph that uses organic carbon for growth, more particularly a saprotroph that obtains nutrients by consuming detritus.

In a further aspect, the present invention provides a plant inoculated with an endophyte as hereinbefore described, said plant comprising an endophyte-free host plant stably infected with said endophyte. Preferably, said plant is a plant with which the endophyte is not naturally associated.

In a preferred embodiment, the plant with which the endophyte is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. Preferably, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a preferred embodiment, the endophyte or plant with which the endophyte is associated may produce one or more compounds that provide beneficial properties such as improved tolerance to water and/or nutrient stress, or improved resistance to pests and/or diseases in the plant with which the fungus is associated. In a preferred embodiment, the beneficial properties include insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a particularly preferred embodiment, the endophyte or plant with which the endophyte is associated may produce an inhibitory compound, such as a nitrification inhibitor, for example a fusicoccane such as brachialactone.

In a preferred embodiment, the host plant may be inoculated with more than one endophyte strain according to the present invention.

Preferably, the plant is an agricultural plant such as a grass species, preferably forage, turf or bioenergy grasses, or a grain crop or industrial crop grass.

The forage, turf or bioenergy grass may be those belonging to the Brachiaria-Urochloa species complex (panic grasses) including Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex, and those belonging to the genera Lolium and Festuca, including L. perenne (perennial ryegrass) and L. arundinaceum (tall fescue) and L. multiflorum (Italian ryegrass).

The grain crop or industrial crop grass may be those belonging to the genus Triticum, including T. aestivum (wheat), those belonging to the genus Hordeum, including H. vulgare (barley), those belonging to the genus Zea, including Z. mays (maize or corn), those belonging to the genus Oryza, including O. sativa (rice), those belonging to the genus Saccharum including S. officinarum (sugarcane), those belonging to the genus Sorghum including S. bicolor (sorghum), those belonging to the genus Panicum, including P. virgatum (switchgrass), and those belonging to the genera Miscanthus, Paspalum, Pennisetum, Poa, Eragrostis and Agrostis.

Preferably, the plant is infected with the endophyte by a method selected from the group consisting of inoculation breeding, crossing, hybridization, transduction, transfection, transformation and/or gene targeting; and combinations thereof.

The endophyte-infected plants may be cultured by known techniques. The person skilled in the art can readily determine appropriate culture conditions depending on the plant to be cultured.

In a further aspect, the present invention provides a plant, plant seed or other plant part derived from a plant of the present invention and stably infected with an endophyte of the present invention. Preferably, the plant, plant seed or other plant part with which the endophyte is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant, plant seed or other plant part. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a particularly preferred embodiment, endophyte or plant with which the endophyte is associated may produce an inhibitory compound, such as a nitrification inhibitor, for example a fusicoccane such as brachialactone.

Preferably, the plant cell, plant, plant seed or other plant part is from a grass, more preferably a forage, turf, bioenergy, grain crop or industrial crop grass.

The forage, turf or bioenergy grass may be those belonging to the Brachiaria-Urochloa species complex (panic grasses), including Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex such as interspecific hybrids between Brachiaria ruziziensis x Brachiaria brizantha, Brachiaria ruziziensis x Brachiaria decumbens, [Brachiaria ruziziensis x Brachiaria decumbens] x Brachiaria brizantha, [Brachiaria ruziziensis x Brachiaria brizantha] x Brachiaria decumbens and those belonging to the genera Lolium and Festuca, including L. perenne (perennial ryegrass) and L. arundinaceum (tall fescue) and L. multiflorum (Italian ryegrass).

The grain crop or industrial crop grass may be those belonging to the genus Triticum, including T. aestivum (wheat), those belonging to the genus Hordeum, including H. vulgare (barley), those belonging to the genus Zea, including Z. mays (maize or corn), those belonging to the genus Oryza, including O. sativa (rice), those belonging to the genus Saccharum including S. officinarum (sugarcane), those belonging to the genus Sorghum including S. bicolor (sorghum), those belonging to the genus Panicum, including P. virgatum (switchgrass), and those belonging to the genera Miscanthus, Paspalum, Pennisetum, Poa, Eragrostis and Agrostis.

By ‘plant cell’ is meant any self-propagating cell bounded by a semi-permeable membrane and containing plastid. Such a cell also required a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

In a further aspect, the present invention provides use of an endophyte as hereinbefore described to produce a plant stably infected with said endophyte. Preferably, the plant with which the endophyte is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a preferred embodiment, the endophyte or plant with which the endophyte is associated may produce one or more compounds that provide beneficial properties such as improved tolerance to water and/or nutrient stress, or improved resistance to pests and/or diseases in the plant with which the fungus is associated. In a preferred embodiment, the beneficial properties include insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a particularly preferred embodiment, the endophyte or plant with which the endophyte is associated may produce an inhibitory compound, such as a nitrification inhibitor, for example a fusicoccane, such as brachialactone.

In another preferred embodiment, the plant with which the endophyte is associated is a forage, turf, bioenergy, grain crop or industrial crop grass as hereinbefore described.

In a further aspect of the present invention, there is provided a method of increasing resistance to pests and/or diseases in a plant, said method including inoculating said plant with an endophyte as hereinbefore described. Preferably, the plant with which the endophyte is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In yet another preferred embodiment, the plant with which the endophyte is associated is a forage, turf, bioenergy, grain crop or industrial crop grass as hereinbefore described.

In another aspect, the present invention provides a method of producing a fusicoccane, said method including

-   -   isolating an endophyte from a plant of the Brachiaria-Urochloa         species complex;     -   growing said endophyte in a suitable culture medium; and     -   recovering one or more organic compounds including the         fusicoccane from endophyte cells, from the culture medium, or         from air space associated with the culture medium or endophyte.

Preferably the endophyte is an endophyte as hereinbefore described.

Preferably, the fusicoccane is compound of formula I:

otherwise known as bracialactone, or a derivative, an isomer and/or a salt thereof.

Preferably, the plant of the Brachiaria-Urochloa species complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, Brachiaria marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota, Urochloa xantholeuca, Brachiaria holosericea, Brachiaria reptans, Brachiaria milliformis, and Brachiaria distachya, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.

Preferably the endophyte is grown in a culture medium including a source of carbohydrates.

The source of carbohydrates may be a starch/sugar-based agar or broth such as potato dextrose agar, potato dextrose broth or half potato dextrose agar or a cereal-based agar or broth such as oatmeal agar or oatmeal broth. Other sources of carbohydrates can include endophyte agar, Murashige and Skoog with 20% sucrose, half V8 juice/half PDA, water agar and yeast malt extract agar.

In a preferred embodiment, the endophyte may be cultured in a culture medium including potato dextrose or oatmeal, for example potato dextrose agar, half potato dextrose agar, oatmeal agar, potato dextrose broth or oatmeal broth. Most preferably, the fungus may be cultured in a culture medium including oatmeal.

The endophyte may be cultured under aerobic or anaerobic conditions.

The endophyte may be cultured for a period of approximately 1 to approximately 100 days, more preferably from approximately 1 to approximately 50 days more preferably from approximately 1 to approximately 10 days.

In a preferred embodiment, the endophyte may be cultured in a bioreactor. By a ‘bioreactor’ is meant a device or system that supports a biologically active environment, such as a vessel in which is carried out a chemical process involving fungi of the present invention and/or products thereof. The chemical process may be aerobic or anaerobic. The bioreactor may have a volume ranging in size from milliliters to cubic metres, for example from approximately 50 millilitres to approximately 50,000 litres. The bioreactor may be operated via batch culture, batch feed culture, perfusion culture or continuous culture, for example continuous culture in a stirred-tank bioreactor. Endophytes cultured in the bioreactor may be suspended or immobilised.

The method includes the step of recovering one or more organic compounds including the fusicoccane from endophyte cells, from the culture medium, or from air space associated with the culture medium or endophyte.

For example, the organic compound(s) may be recovered from intracellular tissues, from the culture medium into which the endophyte may secrete liquids, or from the air space into which the endophyte may secrete vapours.

Vapours may arise directly from the endophyte or from the secreted liquids which transition between vapour and liquid phases.

The step of recovering the organic compound(s) is preferably done by separating cells from the culture medium or capturing vapours associated with the culture medium or endophyte.

Preferably the organic compound(s) is then isolated or purified by a method selected from the group consisting of gas chromatography, liquid chromatography, fractional distillation, cryogenic distillation, membrane separation and absorption chromatography, such as pressure, vacuum or temperature swing adsorption.

By an ‘organic compound’ is meant a chemical compound, the molecules of which contain the element carbon.

In a preferred embodiment, the organic compound may be a hydrocarbon such as a volatile hydrocarbon or a liquid hydrocarbon. Most preferably, the organic compound may be a volatile hydrocarbon.

By a ‘hydrocarbon’ is meant an organic compound comprising the elements carbon and hydrogen.

The term ‘volatile’ in this context is meant an organic compound which can evaporate or sublimate at standard laboratory temperature and pressure. Volatile organic compounds include those with a high vapour pressure, low boiling point and/or low molecular weight.

In a further aspect of the present invention there is provided a method of producing a fusicoccane in a plant of the Brachiaria-Urochloa species complex, said method including:

-   -   providing a plant of the Brachiaria-Urochloa species complex;         and     -   an endophyte, preferably an endophyte as hereinbefore described;     -   infecting said plant with said endophyte to form a symbiota;     -   growing the symbiota in a suitable culture medium, so that the         fusicoccane is produced.

Preferably, the plant of the Brachiaria-Urochloa species complex is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, Brachiaria marlothii, Brachiaria nigropedata, Urochloa dictyoneura, Urochloa oligotricha, Urochloa panicoides, Brachiaria obtusiflora, Brachiaria serrifolia, Urochloa advena, Urochloa arrecta, Urochloa brachyura, Urochloa Urochloa mollis, Urochloa xantholeuca, Urochloa oligotricha, Urochloa panicoides, Urochloa plantaginea, Urochloa platynota, Urochloa xantholeuca, Brachiaria holosericea, Brachiaria reptans, Brachiaria milliformis, and Brachiaria distachya, as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex.

Preferably, the plant is infected with the endophyte by a method selected from the group consisting of inoculation, breeding, crossing, hybridization, transduction, transfection, transformation and/or gene targeting; and combinations thereof.

The endophyte-infected plants may be cultured by known techniques. The person skilled in the art can readily determine appropriate culture conditions depending on the plant to be cultured.

In a further aspect, the present invention provides a plant, plant seed or other plant part derived from a plant produced by the method of the present invention and stably infected with an endophyte of the present invention. Preferably, the plant, plant seed or other plant part with which the endophyte is associated has improved resistance to pests and/or diseases relative to an uninoculated control plant, plant seed or other plant part. In a preferred embodiment, the improved resistance to pests and/or diseases includes insecticidal or insect repellent activity. In a further preferred embodiment, the improved resistance to pests and/or diseases includes antifungal activity.

In a particularly preferred embodiment, the endophyte or plant with which the endophyte is associated may produce an inhibitory compound, such as a nitrification inhibitor, for example a fusicoccane such as brachialactone.

Preferably, the plant cell, plant, plant seed or other plant part is from a grass, more preferably a forage, turf, bioenergy, grain crop or industrial crop grass.

The forage, turf or bioenergy grass may be those belonging to the Brachiaria-Urochloa species complex (panic grasses), including Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis as well as interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex such as interspecific hybrids between Brachiaria ruziziensis x Brachiaria brizantha, Brachiaria ruziziensis x Brachiaria decumbens, [Brachiaria ruziziensis x Brachiaria decumbens] x Brachiaria brizantha, [Brachiaria ruziziensis x Brachiaria brizantha] x Brachiaria decumbens and those belonging to the genera Lolium and Festuca, including L. perenne (perennial ryegrass) and L. arundinaceum (tall fescue) and L. multiflorum (Italian ryegrass).

The grain crop or industrial crop grass may be those belonging to the genus Triticum, including T. aestivum (wheat), those belonging to the genus Hordeum, including H. vulgare (barley), those belonging to the genus Zea, including Z. mays (maize or corn), those belonging to the genus Oryza, including O. sativa (rice), those belonging to the genus Saccharum including S. officinarum (sugarcane), those belonging to the genus Sorghum including S. bicolor (sorghum), those belonging to the genus Panicum, including P. virgatum (switchgrass), and those belonging to the genera Miscanthus, Paspalum, Pennisetum, Poa, Eragrostis and Agrostis.

In another aspect, the present invention provides a method of inoculating a plant of the Brachiaria-Urochloa species complex with one or more endophytes, said method including

-   -   providing sterilised seed of the plant of the         Brachiaria-Urochloa species complex;     -   germinating the seed under aseptic conditions to produce host         plants that are substantially free of microbial organisms;     -   inoculating the host plants with the one or more endophytes.

While Applicant does not wish to be restricted by theory, it is thought that conducting the method of the present invention under aseptic conditions ensures endophytes are inoculated into host plants that are substantially free of microbial organisms, thereby facilitating a high frequency of successful inoculation. Further, it is thought that the use of different media prior to inoculation to allow the host plant to establish, such as root growth promoting media, also facilitates high inoculation frequency. The use of a sterile environment also enables analysis of the microbiome without contamination.

For example the inoculation frequency may be between approximately 25% and approximately 100%, more preferably between approximately 50% and approximately 100%, even more preferably between approximately 75% and approximately 100%. The inoculation frequency may be higher than conventional methods.

Preferably, the plant of the Brachiaria-Urochloa species complex is of a species as hereinbefore described.

Preferably said one or more endophytes are selected from the endophytes as hereinbefore described. The one or more endophytes may be bacterial or fungal or a mixture thereof. In a preferred embodiment, the step of germinating the seed under aseptic conditions to produce host plants may include growing the germinated seed on shoot multiplication medium such as M3B and root multiplication medium such as MS+NAA. Preferably, the germinated seed may be grown on shoot multiplication medium, splitting the resulting shoots into single tillers and then transferring them to root multiplication medium. The single tillers may be grown on the root multiplication medium for approximately 1 to approximately 6 weeks, more preferably approximately 2 to approximately 3 weeks, to promote root growth. The resulting plantlets may again be split into single tillers for endophyte inoculation.

In a preferred embodiment, the step of inoculating the host plants with the one or more endophytes may include removal of the outer sheath to reveal shoot initial, creation of a wound in the shoot meristem and inoculation into the wound.

In a preferred embodiment, the method may include the further step of retaining the plantlets on sterile media following inoculation, preferably for a period of approximately 1 to approximately 6 weeks, more preferably approximately 2 to approximately 3 weeks.

In a preferred embodiment, the method may include the still further step of transferring the inoculated plants thus produced to soil or similar medium for further growth, for example under glasshouse conditions.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows the incidence of endophyte isolates in Brachiaria species based on rDNA sequence analysis of the internal transcribed spacer (ITS) and 18S coding regions. Brachiaria endophyte isolates are genetically diverse, representing at least 10 distinct taxonomic groups.

FIG. 2 shows a bootstrap consensus tree generated through neighbour-joining analysis of the ITS region from fungal endophyte isolates derived from Brachiaria-Urochloa accessions (left hand side) and the identification of the presence/absence of selected isolated and culturable fungal endophyte strains in the microbiomes of 5 Brachiaria-Urochloa species (right hand side) (Bb—B. brizantha; Bh—B. humidicola; Bd—B. decumbens; Um—U. mosambicensis; Br—B. ruziensis) based on the ITS sequence.

FIG. 2A is an enlarged bootstrap consensus tree of FIG. 2.

FIG. 3 shows an LC(ESI)-MS mass spectra identifying brachialactone, displaying extracted ion chromatogram at RT: 8.91-8.98 minutes.

FIG. 4 shows MS fragmentation brachialactone fragments (333.2059, 315.1964, 271.2068) at RT 8.93 minutes of the LC(ESI)-MS mass spectra of FIG. 3.

FIG. 5 shows the structure of brachialactone and the structures of the fragments responsible for the fragmentation of FIG. 4.

FIG. 6 shows consecutive stages of an optimised endophyte inoculation procedure for Brachiaria-Urochloa. A. Microbe-free donor plantlets grown on shoot multiplication media (M3B) under sterile conditions; B. Donor shoots split into single tillers and transferred to root multiplication media (MS+NAA); C. Single tillers grown for 2-3 weeks to promote root growth prior to splitting plantlets into single tillers, removal of the outer sheath to reveal shoot initial, transferal to water agar and inoculation into a small cut is made across the shoot meristem; D. Inoculated plantlets retained on ½ MS media for 2 weeks; E. Plantlets after transfer to soil and growth under glasshouse conditions for 8 weeks.

FIG. 7 shows a flow chart describing a method for profiling the seed associated bacterial and fungal microbiome. The accompanying photographs show each stage of the method of enriching for bacterial and fungal DNA from 50 g seed from one accession. A. Air drying the seeds prior to grinding; B. Collecting the 100% ethanol wash. The supernatant contains the seed associated endophytic microbiome; C. Vials during evaporation of ethanol; D. Analysis of extracted DNA from each of the 10 Accessions (columns 1 to 10) to determine presence of fungal DNA. E. Analysis of extracted DNA to determine presence of bacterial DNA.

DETAILED DESCRIPTION OF THE EMBODIMENTS Example 1—Endophytic Microbial Profile of Brachiaria-Urochloa Species

The endophytic microbiomes of five Brachiaria-Urochloa species were profiled using metagenomics. Species included B. brizantha, B. humidicola, B. ruziziensis, B. decumbens and U. mosambicensis. A total of three plants were profiled per species. Three organs were profiled from each plant (roots, stem and leaves). A total of two replicates were prepared per organ, per plant. Plant material (approximately 100 mg) was surface sterilised by soaking in 70% ethanol for 30 seconds, followed by 4.2% NaOCl (bleach) for 2 minutes, and then rinsed three to five times in sterile MilliQ water to ensure the sterilant had been completely removed. Samples were freeze-dried for 48 hours at −58° C. and 0.014 mBar. DNA was extracted using the Qiagen DNeasy plant mini kit according to manufacturer's instructions. Endophytic bacteria and fungi were evaluated in the metagenomics analyses using the universal PCR primers 515f and 806r for profiling the bacterial microbiome (V4 region of the 16S rDNA gene, approx. 350 base pairs), and 58A2F and ITS4 for the fungal microbiome (ITS2 region of the rDNA genes, approx. 400 base pairs), with associated Illumina adapters. Paired end libraries were prepared and loaded according to the corresponding Illumina user guide. Metagenomic sequence data was quality trimmed and paired using PANDSEQ to create operational taxonomic units (OTU), which were aligned against the GreenGenes bacterial database and the UNITE fungal database to assign taxonomy (OTU: 97% sequence identity, e-value <10e-110). The number of sequences associated with OTUs was calculated across all samples, and normalised as a percentage.

Total Microbial Diversity Across Brachiaria-Urochloa

A total of 361 bacterial operational taxonomic units (OTUs) were identified across all Brachiaria-Urochloa species, comprising 25 bacterial Phyla, 56 Classes, 121 Families and 170 Genera (including candidate taxonomic groups) (Table 1). The analyses identified the core microbiome (OTUs found across all Brachiaria-Urochloa species) and the unique microbiome (OTUs associated with specific Brachiaria-Urochloa species), along with bacterial OTUs only associated with Brachiaria-Urochloa species known to produce brachialactone (B. humidicola and B. ruziziensis) (Table 1). The analyses also provided cross validation of the presence of endophytes isolated from these Brachiaria-Urochloa species.

In addition, 84 fungal OTUs were identified, comprising 5 Phyla, 14 Classes, 32 Families and 44 Genera (Table 2). The analyses identified the core microbiome (OTUs found across all Brachiaria-Urochloa species) and the unique microbiome (OTUs associated with specific Brachiaria-Urochloa species), along with fungal OTUs only associated with Brachiaria-Urochloa species known to produce brachialactone (B. humidicola and B. ruziziensis) (Table 2). The analyses also provide cross validation of the presence of endophytes isolated from these Brachiaria-Urochloa species.

TABLE 1 Endophytic bacterial OTUs found within the leaves, root and stem of five Brachiaria-Urochloa species (361 OTUs), including identification of the core microbiome, the unique microbiome, and bacterial OTUs only associated with Brachiaria-Urochloa species known to produce brachialactone (Brach - B. humidicola and B. ruziziensis). Examples of the bacterial microbial diversity in Brachiaria-Urochloa species is represented within B. humidicola (Bh) and B. decumbens (Bd). Endophytic bacterial OTU Leaves Stems Roots Core Unique Isolated Brach Bh Bd 1 Bacteria sp 1 Y Y Y Y Y Y 2 Enterobacteriaceae sp Y Y Y Y Y Y 3 Rhodospirillaceae sp Y Y Y Y Y 4 Comamonadaceae sp Y Y Y Y Y 5 Agrobacterium sp Y Y Y Y Y 6 Alicyclobacillaceae sp Y Y Y Y 7 Sphingobacteriaceae sp Y Y Y Y Y 8 Mycobacterium sp Y Y Y Y 9 Bacillus sp Y Y Y Y 10 Chitinophagaceae sp Y Y Y Y Y 11 Pseudomonas sp Y Y Y Y Y 12 Caulobacteraceae sp Y Y Y Y Y 13 Rhodoplanes sp Y Y Y Y Y 14 Rhizobium sp Y Y Y Y Y 15 Rhizobiales sp Y Y Y Y 16 Janthinobacterium sp Y Y Y Y 17 Cellulomonas sp Y Y Y Y 18 Herbaspirillum sp Y Y Y Y Y 19 Flavobacterium sp Y Y Y Y 20 Xanthomonadaceae sp Y Y Y Y 21 Solirubrobacterales sp Y Y Y Y 22 Dyella spginsengisoli Y Y Y Y 23 Betaproteobacteria sp 1 Y Y Y Y 24 Opitutus sp Y Y Y Y Y 25 Aquicella sp Y Y Y Y 26 Candidatus_ Y Y Y Y Xiphinematobacter sp 27 Microbacterium sp Y Y Y Y 28 Flavobacterium Y Y Y Y Y spsuccinicans 29 Kiloniellales sp Y Y Y Y Y 30 Alphaproteobacteria sp 1 Y Y Y Y Y 31 Devosia sp Y Y Y Y 32 Planctomyces sp Y Y Y Y Y 33 Sphingomonadaceae sp Y Y Y Y Y 34 Isosphaeraceae sp Y Y Y Y 35 Asticcacaulis Y Y Y Y spbiprosthecium 36 Pirellulaceae sp 1 Y Y Y Y Y 37 Myxococcales sp 1 Y Y Y Y Y 38 Gammaproteobacteria sp Y Y Y Y 39 Mycobacterium spvaccae Y Y Y Y 40 Chitinophaga sp Y Y Y Y Y 41 Dechloromonas sp Y Y Y Y Y 42 Streptomyces sp Y Y Y Y 43 Desulfosporosinus Y Y Y Y spmeridiei 44 Rhodocyclaceae sp Y Y Y Y 45 Novosphingobium sp Y Y Y Y 46 Oxalobacteraceae sp Y Y Y Y 47 Opitutaceae sp Y Y Y Y 48 Hyphomicrobium sp Y Y Y Y 49 Legionellaceae sp Y Y Y Y 50 Rhodanobacter sp Y Y Y Y 51 Sinobacteraceae sp Y Y Y Y Y 52 Sphingobium sp Y Y Y Y 53 Nocardioidaceae sp Y Y Y Y 54 Azospirillum sp Y Y Y Y Y 55 Acidobacteria sp 1 Y Y Y Y 56 Alphaproteobacteria sp 2 Y Y Y Y 57 Klebsiella sp Y Y Y Y 58 Pleomorphomonas Y Y Y Y sporyzae 59 Rhizobiaceae sp Y Y Y Y 60 Bacillus spginsengihumi Y Y Y Y 61 Rhodobacteraceae sp Y Y Y Y 62 Cytophagaceae sp Y Y Y Y Y 63 Bradyrhizobium sp Y Y Y Y 64 Methylotenera spmobilis Y Y Y Y 65 Microbacterium Y Y Y spchocolatum 66 Acidobacteria sp 2 Y Y Y Y 67 Rhodobacter sp Y Y 68 Coxiellaceae sp Y Y Y Y 69 Acidimicrobiales sp 1 Y Y Y Y 70 Mesorhizobium sp Y Y Y Y 71 Cellvibrio sp Y Y Y Y 72 Methylibium sp Y Y Y Y 73 Rubrivivax spgelatinosus Y Y Y Y 74 Clostridium sp Y Y Y Y 75 Erythrobacteraceae sp Y Y Y Y 76 Pedosphaerales sp 1 Y Y Y Y Y 77 Microbacteriaceae sp Y Y Y Y 78 Thermomicrobia sp Y Y Y Y 79 Hyphomicrobiaceae sp Y Y Y Y 80 Bacteria sp 2 Y Y Y Y 81 Sphingobacteriales sp Y Y Y Y 82 Bradyrhizobiaceae sp Y Y Y Y 83 Acetobacteraceae sp Y Y Y Y Y 84 Phaeospirillum spfulvum Y Y 85 Legionella sp Y Y Y Y 86 Dyella sp Y Y Y Y 87 Gemmata sp Y Y Y Y 88 Hydrogenophaga sp Y Y Y Y 89 Betaproteobacteria sp 2 Y Y Y Y Y 90 Rhodanobacter Y Y Y Y splindaniclasticus 91 Pedobacter sp Y Y Y Y 92 Asticcacaulis sp Y Y Y Y 93 Magnetospirillum sp Y Y Y Y 94 Prosthecobacter sp Y Y Y Y Y 95 Paenibacillus sp Y Y Y Y 96 Sphingomonas spwittichii Y Y Y Y 97 Methylophilaceae sp Y Y Y Y 98 Sphingomonas sp Y Y Y Y 99 Chryseobacterium sp Y Y 100 Chloroflexi sp 1 Y Y Y 101 Propionivibrio sp Y Y Y Y 102 Phenylobacterium sp Y Y Y Y 103 Delftia sp Y Y Y Y 104 Bacillales sp Y Y Y Y 105 Erwinia sp Y Y Y Y 106 Limnohabitans sp Y Y Y Y 107 Cohnella sp Y Y Y Y 108 Denitrobacter sp Y Y Y Y 109 Kaistia sp Y Y Y Y 110 Dyadobacter sp Y Y 111 Alphaproteobacteria sp 3 Y Y 112 Sphingomonas Y Y Y spazotifigens 113 Actinomycetales sp Y Y Y Y 114 Pirellulaceae sp 2 Y Y Y Y 115 Fluviicola sp Y Y Y Y 116 Shinella sp Y Y Y Y 117 Spirochaeta spaurantia Y Y Y 118 Brucellaceae sp Y Y 119 Agrobacterium spsullae Y Y Y Y 120 Fibrobacteria sp Y Y Y Y Y 121 Alteromonadales sp 1 Y Y Y Y 122 Nakamurellaceae sp Y Y Y Y 123 Alcaligenaceae sp Y Y Y Y 124 Desulfovibrio sp Y Y Y Y 125 Sediminibacterium sp Y Y Y Y 126 Cryocola sp Y Y 127 Sphingopyxis sp Y Y 128 Burkholderia sp Y Y Y Y 129 Gaiellaceae sp Y Y Y 130 Niastella sp Y Y Y 131 Rathayibacter sp Y Y 132 Pandoraea sp Y Y Y 133 Burkholderiales sp Y Y Y Y 134 Thermomonas sp Y Y 135 Novosphingobium Y Y Y Y spcapsulatum 136 Pseudomonas Y Y Y Y spnitroreducens 137 Lachnospiraceae sp Y Y 138 Bacteria sp 3 Y Y Y Y 139 Caldilineaceae sp Y Y Y 140 Micrococcaceae sp Y Y 141 Bacteria sp 4 Y Y Y 142 Geobacillus sp Y Y Y 143 Salinibacterium sp Y Y 144 Rickettsiales sp Y Y Y Y 145 Cyanobacteria sp 1 Y Y Y Y 146 Hyphomonadaceae sp Y Y Y Y 147 Salinispora sptropica Y Y Y 148 Bdellovibrio sp Y Y 149 Caulobacter sp Y Y Y Y 150 Salinispora sp Y Y Y Y 151 Sulfurospirillum sp Y Y 152 Uliginosibacterium sp Y Y Y Y 153 Bdellovibrio Y Y Y spbacteriovorus 154 Chloroflexi sp 2 Y Y 155 Ruminococcaceae sp Y Y 156 Anaerolineae sp 1 Y Y Y Y 157 Kyrpidia sp Y Y Y Y 158 Rhodoferax sp Y Y 159 Aurantimonadaceae sp Y Y 160 Curtobacterium sp Y Y Y 161 Cupriavidus sp Y Y Y Y 162 Nocardioides sp Y Y Y 163 Legionellales sp Y Y 164 Coprococcus sp Y Y Y Y 165 Pseudomonadaceae sp Y 166 Emticicia sp Y Y Y Y 167 Procabacteriaceae sp Y Y 168 Aeromonadaceae sp Y Y 169 Cytophaga sp Y Y 170 Haliangiaceae sp Y Y 171 Chloroflexi sp 3 Y Y Y 172 Gemmatimonadetes sp 1 Y Y Y 173 Betaproteobacteria sp 3 Y Y Y 174 Demequina sp Y Y 175 Cyanobacteria sp 2 Y Y 176 Moraxellaceae sp Y 177 Chlorobi sp 1 Y Y Y 178 Bacteriovoracaceae sp Y Y 179 Paludibactersp Y Y Y 180 Burkholderia spbryophila Y Y Y 181 Porphyromonadaceae sp Y Y Y 182 Leptothrix sp Y Y Y 183 Gemmataceae sp Y Y Y 184 Aminobacter sp Y Y Y 185 Desulfovibrio spmexicanus Y Y 186 Flavihumibacter sp Y Y 187 Ramlibacter sp Y Y 188 Neisseriaceae sp Y Y 189 Asteroleplasma sp Y Y Y 190 Edaphobacter spmodestum Y Y 191 Verrucomicrobiaceae sp Y Y Y 192 Patulibacteraceae sp Y Y Y 193 Arthrospira sp Y Y 194 Achromobacter sp Y Y 195 Desulfobulbus sp Y Y 196 Pelomonas sp Y Y 197 Zoogloea sp Y Y 198 Desulfovibrio spputealis Y Y Y Y 199 Salmonella spenterica Y Y 200 Acidimicrobiales sp 2 Y Y Y 201 Bacteria sp 5 Y Y 202 Phyllobacteriaceae sp Y Y 203 Dokdonella sp Y Y 204 Pedosphaerales sp 2 Y Y 205 Variovorax spparadoxus Y Y Y 206 Armatimonadetes sp Y Y 207 Bacteroidales sp Y Y 208 Chloroflexi sp 4 Y Y 209 Geobacter sp Y Y Y 210 Deltaproteobacteria sp 1 Y Y 211 Sulfuricurvum spkujiense Y Y 212 Phaeospirillum sp Y Y 213 Tatlockia sp Y Y 214 Terriglobus sp Y Y Y 215 Pelosinus sp Y Y 216 Thermomonas spfusca Y Y Y Y 217 Gemmata spobscuriglobus Y 218 Telmatospirillum sp Y Y 219 Luteibacter sprhizovicinus Y Y Y 220 Acidisoma sp Y Y Y 221 Fimbriimonas sp Y Y 222 Janibactersp Y Y 223 Anaerolineae sp 2 Y Y 224 Chloroflexi sp 5 Y Y 225 Thermoanaerobacterium Y Y Y spsaccharolyticum 226 Luteolibacter sp Y 227 Leadbetterella sp Y Y 228 Afifella sp Y Y Y 229 Microthrixaceae sp Y Y 230 Chlorobi sp 2 Y Y 231 Ancylobacter sp Y 232 Steroidobacter sp Y Y Y Y 233 Singulisphaera sp Y Y 234 Luteimonas sp Y Y 235 Gemmatimonadetes sp 2 Y Y Y 236 Bosea spgenosp. Y Y 237 Salinibacterium Y Y spamurskyense 238 Pedosphaerales sp 3 Y Y 239 Phycicoccus sp Y Y 240 Spirosoma sp Y Y 241 Chromatiales sp Y Bd Y 242 Rathayibacter spcaricis Y Y 243 Treponema sp Y Y Y 244 Holophagaceae sp Y Y 245 Anaerovorax sp Y Y 246 Desulfobulbaceae sp Y Y 247 Reichenbachiella sp Y 248 Nannocystis sp Y Ur Y 249 Polyangiaceae sp Y Y 250 Haererehalobacter Y Y spsalaria 251 Streptomyces splanatus Y Y 252 Aquitalea spmagnusonii Y Y 253 Erwinia spsoli Y Y 254 Trabulsiella sp Y Y 255 Pilimelia sp Y Y Y 256 Clostridia sp Y Y Y 257 Ammoniphilus sp Y Y 258 Paenibacillaceae sp Y Y 259 Streptosporangiaceae sp Y 260 Methylocystaceae sp Y Y 261 Solibacterales sp Y Y 262 Acidobacteria sp 3 Y Y 263 Frankiaceae sp Y Y 264 Acidovorax spdelafieldii Y 265 Piscirickettsiaceae sp Y Y 266 Candidatus_Solibacter sp Y Bd Y 267 Parvibaculum sp Y Y Y 268 Betaproteobacteria sp 4 Y Y Y 269 Acidobacteria sp 4 Y Bb 270 Cellulomonas spuda Y Y 271 Chloroflexi sp 6 Y Y Y Y 272 Brevibacillus spreuszeri Y Y Y 273 Blastomonas sp Y Y 274 Bacteria sp 6 Y Y 275 Streptomycetaceae sp Y Y 276 Sphingomonas Y Y spechinoides 277 Polaromonas sp Y Y 278 Cellulomonadaceae sp Y Bd Y 279 Armatimonadia sp Y Ur Y 280 Cyanobacteria sp 3 Y Bb 281 Comamonas sp Y Ur Y 282 Gracilibacteraceae sp Y Ur Y 283 Cenarchaeaceae sp Y Y 284 Acidovorax sp Y 285 Janthinobacterium Y Ur Y splividum 286 Acinetobacter sp Y Y 287 Ruminococcus sp Y Bd Y 288 Simplicispira sp Y Ur Y 289 Rheinheimera sp Y Um 290 Pseudomonas spstutzeri Y 291 Acidobacteriaceae sp Y Y Y 292 Kouleothrixaceae sp Y Y 293 Azospira sp Y Ur Y 294 Chromatiaceae sp Y Y 295 Pseudomonas Y Ur Y spviridiflava 296 Staphylococcus spaureus Y Y 297 Alteromonadaceae sp Y Y 298 Aeromicrobium sp Y Bb 299 Chthoniobacter sp Y Ur Y 300 Saprospiraceae sp Y Bb 301 Bacillus spcoagulans Y Bd Y 302 Frankia sp Y Bb 303 Kineosporiaceae sp Y Bd Y 304 Alicyclobacillus sp Y Ur Y 305 Sporolactobacillaceae sp Y Ur Y 306 Pedosphaerales sp 4 Y Bd Y 307 Nocardia sp Y Bd Y 308 Planctomycetes sp Y Bh Y Y 309 Syntrophobacteraceae sp Y Bb 310 Bacteria sp 7 Y Bd Y 311 Xanthobacteraceae sp Y Bb 312 Saprospirales sp Y Ur Y 313 Geobacillus Y Ur Y spthermodenitrificans 314 Paenibacillus Y Ur Y spchondroitinus 315 Mycoplana sp Y Bd Y 316 Corynebacterium sp Y Bb 317 Microbacterium spaurum Y Bd Y 318 Nostocaceae sp Y Bb 319 Thermoanaerobacterium Y Ur Y sp 320 Peptococcaceae sp Y Bb 321 Clostridiales sp Y Ur Y 322 Ochrobactrum sp Y Bd Y 323 Myxococcales sp 2 Y Bb 324 Pseudomonas Y Ur Y spalcaligenes 325 Methanobacterium sp Y Bh Y Y 326 Methylophilales sp Y Um 327 Enterobacter sp Y Bd Y 328 Niabella sp Y Ur Y 329 Bacillaceae sp Y Um 330 Balneimonas sp Y Bb 331 Schlegelella sp Y Ur Y 332 Deltaproteobacteria sp 2 Y Bb 333 Bacteria sp 8 Y Ur Y 334 Chthoniobacteraceae sp Y Bd Y 335 Acidobacteria sp 5 Y Bd Y 336 Inquilinus sp Y Bb 337 Myxococcaceae sp Y Bd Y 338 Prosthecobacter Y Bd Y spdebontii 339 Dermacoccus sp Y Bd Y 340 Jonesiaceae sp Y Ur Y 341 Actinoplanes sp Y Bd Y 342 Clostridium spbowmanii Y Um 343 Bacteria sp 9 Y Bd Y 344 Chromobacterium sp Y Ur Y 345 Tolumonas sp Y Um 346 Alteromonadales sp 2 Y Bd Y 347 Bacteria sp 10 Y Ur Y 348 Corynebacterium Y Bh Y Y spkroppenstedtii 349 Cloacibacterium sp Y Bd Y 350 Thermogemmatispora sp Y Bh Y Y 351 Staphylococcus Y Bd Y spepidermidis 352 Sporomusa sp Y Bb 353 Azovibrio sp Y Bb 354 Alteromonadales sp 3 Y Ur Y 355 Erwinia spdispersa Y Ur Y 356 Acinetobacter spjohnsonii Y Ur Y 357 Bacteroides sp Y Bb 358 Anaerococcus sp Y Bb 359 Peptoniphilus sp Y Bb 360 Acidocella sp Y Bh Y Y 361 Desulfovibrionaceae sp Y Ur Y 0

TABLE 2 Endophytic fungal OTUs found within the leaves, root and stem of five Brachiaria- Urochloa species (84 OTUs), including identification of the core microbiome, the unique microbiome, and fungal OTUs only associated with Brachiaria-Urochloa species known to produce brachialactone (Brach - B. humidicola and B. ruziziensis). Examples of the fungal microbial diversity in Brachiaria species is represented within B. humidicola (Bh) and B. decumbens (Bd). Endophytic fungal OTU Leaves Stems Roots Core Unique Isolated Brach Bh Bd 1 Dothideomycetes sp Y Y Y Y Y Y 2 Uncultured Fungal sp 1 Y Y Y Y Y Y Y (Microdochium bolleyi) 3 Fusarium proliferatum Y Y Y Y Y Y 4 Lecythophora sp Y Y Y Y 5 Chaetosphaeriales sp Y Y Y Y Y Y 6 Sarocladium strictum Y Y Y Y Y Y 7 Chaetomium sp 1 Y Y Y Y 8 Uncultured Glomus sp Y Y Y Y 9 Sordariomycetes sp Y Y Y Y 10 Uncultured Fungal sp (soil) Y Y Y Y 11 Hypocrea sp Y Y Y Y Y 12 Coniochaeta sp Y Y Y Y 13 Microsphaeropsis arundinis Y Y Y Y Y 14 Uncultured Sebacina sp 1 Y Y Y Y 15 Chrysosporium sp 1 Y Y Y 16 Acremonium sp Y Y Y 17 Podospora sp 1 Y Y Y 18 Fusarium sp Y Y Y 19 Fusarium oxysporum f Y Y Y sp ciceris 20 Uncultured Ascomycota sp Y Y 21 Rhizophagus sp Y Y Y 22 Cryptococcus laurentii Y Y 23 Flagelloscypha minutissima Y Y 24 Podospora communis Y Y 25 Cladosporium cladosporioides Y Y Y 26 Uncultured Rhizophagus sp Y Y Y 27 Exophiala cancerae Y Y 28 Uncultured Coprinus sp Y Y 29 Uncultured Olpidium sp Y Y Y 30 Rhizophagus irregularis Y Y Y 31 Uncultured Fungal sp 2 Y 32 Myrmecridium schulzeri Y Y Y 33 Uncultured Y Urediniomycete sp 34 Rhizophagus irregularis Y Y Y DAOM 181602 35 Paraglomus brasilianum Y Y Y 36 Fungal sp 1 Y 37 Parascedosporium putredinis Y Y Y 38 Uncultured Sebacina sp 2 Y Y Y 39 Uncultured Acaulospora sp Y Y Y 40 Chaetomium thermophilum Y Y 41 Candida tropicalis Y Y 42 Conlarium sp Y Y 43 Arthrobotrys sp Y Y 44 Candida sp Y Y 45 Trichoderma harzianum Y Y Y 46 Pseudeurotium sp Y Ur Y 47 Chaetomium sp 2 Y 48 Doratomyces sp Y Y 49 Zopfiella marina Y Um 50 Monographella cucumerina Y Bb Y 51 Clitopilus scyphoides Y Y 52 Acephala sp Y Y 53 Trichurus sp Y Y Y 54 Corynascus sp Y Ur Y 55 Uncultured Archaeospora sp Y Ur Y 56 Trichoderma asperellum Y Bb Y 57 Gibberella fujikuroi Y Um 58 Fusarium sp Y Ur Y 59 Fungal sp 2 Y Bh Y Y 60 Fusarium oxysporum Y Bh Y Y 61 Peziza ostracoderma Y Bb Y 62 Pseudallescheria boydii Y Um 63 Fungal sp 3 Y Um 64 Pseudogymnoascus sp Y Bh Y Y Y 65 Claroideoglomus sp Y Bh Y Y 66 Leptosphaeria sp Y Bh Y Y 67 Uncultured Y Bh Y Y Herpotrichiellaceae 68 Haptocillium sinense Y Bd Y 69 Piriformospora indica Y Um 70 Fungal sp 4 Y Um 71 Exophiala sp Y Bh Y Y 72 Neotyphodium sp FaTG 2 Y Bd Y 73 Uncultured Trichoderma sp Y Bb Y 74 Podospora sp 2 Y Bh Y Y 75 Meira sp Y Bh Y Y 76 Leptosphaerulina chartarum Y Ur Y 77 Chrysosporium sp 2 Y Bb Y 78 llyonectria sp Y Bh Y Y 79 Gibberella intricans Y Um 80 Ophiostoma stenoceras Y Bh Y Y 81 Microbotryomycetes sp Y Um 82 Cryptococcus podzolicus Y Bb Y 83 Fungal sp (endophyte) Y Bb Y 84 Fungal sp 5 Y Bh Y Y

Microbial Diversity Between Plant Organs of Brachiaria-Urochloa

Microbial diversity was greatest in the roots of Brachiaria-Urochloa species accounting for 359 bacterial species (99.4%) and 83 fungal species (98.8%) (Tables 1 and 2). The microbial diversity in the stem and leaf was significantly lower than the roots, accounting for 5-20 bacterial or fungal OTUs.

Microbial Diversity Across Brachiaria-Urochloa Species

The core microbiome consisted of 130 bacterial OTUs and 14 fungal OTUs (Tables 1, 2, and 3). The core bacterial microbiome contained a diverse array of taxa, while the core fungal microbiome predominantly contained Sordariomycetes species (8). The OTUs associated with the core microbiome were also the most abundant OTUs across all Brachiaria-Urochloa species. The number of OTUs unique to Brachiaria-Urochloa species ranged from 5 to 27 for bacteria and 2 to 12 for fungi, and were predominantly found in low abundance in their respective species.

TABLE 3 The core and unique microbiome associated with Brachiaria-Urochloa species B. B. B. U. B. brizantha decumbens humidicola mosambicensis ruziziensis Core Bacteria 130 Fungi 14 Unique Bacteria 19 23 5 5 27 Fungi 7 2 12 8 5 Microbial Species Associated with B. humidicola and B. decumbens

The number of bacterial and fungal OTUs associated with B. humidicola was 189 and 48 respectively. B. humidicola had the highest fungal diversity, approximately 15% higher than any other Brachiaria-Urochloa species. Conversely, B. humidicola had the second lowest bacterial diversity, approximately 31% lower than B. decumbens (highest bacterial diversity). As with all other Brachiaria-Urochloa species the greatest microbial diversity was observed in the roots, while there was very low microbial diversity in the stems and leaves.

The number of bacterial and fungal OTUs associated with B. decumbens was 276 and 37 respectively. B. decumbens had the highest bacterial diversity, approximately 3 to 33% higher than any other Brachiaria-Urochloa species. Conversely, B. decumbens had the second lowest fungal diversity, approximately 23% lower than B. humidicola. As with all other Brachiaria-Urochloa species the greatest microbial diversity was observed in the roots, while there was very low microbial diversity in the stems and leaves.

The top five fungal and bacterial OTUs associated with both B. humidicola and B. decumbens show sequence homology to isolates from NCBI that have been predominantly been identified as endophytes, including endophytes of other Poaceae species (e.g. Oryzae sativa, Triticum aestivum), mycorrhizae (e.g. Glomus species) or rhizobacteria (Rhizobiales species). The fungal pathogen Fusarium proliferatum was also present, which is a seed-borne pathogen of a range of agricultural crop species (Tables 4 and 5).

TABLE 4 Examples of the most abundant fungal OTUs associated with Brachiaria-Urochloa species, and their corresponding NCBI top Blastn hit (accession number, e-value, isolation source, and endophytic origin - E+/−). OTU (Unite Hit) NCBI Hit E+/− Isolation Source Accession E−Value 1 Dothidiomycetes Fungal sp. + Trillium GU479902.1 7.0E−149 sp. tschonoskii Fungal endophyte + Holcus lanatus FN394695.1 7.0E−149 2 Uncultured Microdochium + Triticum KC989068.1 3.0E−163 Fungal sp bolleyi aestivum Uncultured root- + Sporobolus FJ362153.1 1.0E−141 associated fungus cryptandrus 3 Chaetosphaeriales Chaetosphaeriales + Populus KF428394.1 2.0E−149 sp. sp. trichocarpa Chaetosphaeriaceae + Populus JX244066.1 2.0E−149 deltoides 4 Fusarium Fusarium + Dendrobium KM023784.1 2.0E−159 proliferatum proliferatum sp. Fusarium sp. + Saccharum KF293339.1 2.0E−159 officinarum 8 Uncultured Uncultured Glomus + Allium cepa L. AM992800.1 0.0E+00  Glomus sp. Uncultured + Sequoiadendron HQ895815.2 8.0E−179 Glomeromycota giganteum

TABLE 5 Examples of the most abundant bacterial OTUs associated with Brachiaria species, and their corresponding NCBI top Blastn hit (accession number, e-value, isolation source, and endophytic origin - E+/−). OTU Isolation (GreenGenes Hit) NCBI Hit E+/− Source Accession E−Value 1 Enterobacteriaceae Enterobacter − Oryza sativa NR_125587.1 2.0E−132 sp oryziphilus Kosakonia + Saccharum NR_118333.1 4.0E−129 sacchari officinarum 2 Pseudomonas sp Pseudomonas − Oryza sativa NR_074659.1 2.0E−132 fulva soil Pseudomonas − — NR_074599.1 8.0E−131 protegens Pf-5 Pseudomonas − Soil NR_102854.1 2.0E−127 entomophila 3 Agrobacterium sp Agrobacterium − — NR_041396.1 2.0E−132 tumefaciens Rhizobium + Astragalus NR_117440.1 4.0E−129 vignae dahuricus Rhizobium + Lemna NR_114340.1 8.0E−126 paknamense aequinoctialis 4 Comamonadaceae Ottowia − Coking NR_125656.1 8.0E−131 sp shaoguanensis wastewater Comamonas − — NR_114013.1 4.0E−129 granuli Comamonas − Activated NR_102841.1 2.0E−127 testosteroni sludge 5 Herbaspirillum sp Herbaspirillum + Miscanthus NR_025353.1 2.00E−132 frisingense sacchariflorus Herbaspirillum − — NR_024698.1 2.00E−132 huttiense Oxalicibacterium − Soil NR_112833.1 8.00E−126 horti Microbial Diversity Associated with Brachialactone Producing Brachiaria-Urochloa Species

A total of 45 bacterial and 29 fungal OTUs were identified only in the Brachiaria-Urochloa species found to produce brachialactone, B. humidicola and/or B. ruziziensis (Tables 1 and 2). The OTUs associated with brachialactone producing Brachiaria-Urochloa species represent a range of diverse bacterial and fungal taxa.

Example 2—Metagenome Analysis of the Brachiaria Microbiome Determines the Host Range of Fungal Endophytes of Brachiaria-Urochloa Grasses

A total of 97 fungal endophyte isolates derived from 11 Brachiaria-Urochloa species were identified in a global study of 281 accessions from 23 countries. The internal transcribed spacer ITS sequence was used for further characterisation. The entire region of nuclear ribosomal DNA which comprises both internal transcribed spacers ITS1 and ITS2 and the 5.8S subunit was PCR-amplified using primers ITS5 and ITS4 (White et al. 1990). Purified PCR amplification products were sequenced using Sanger sequencing technology. Isolated subcultured endophytes were then grouped based on ITS sequence identity. Ribosomal DNA (rDNA) sequence analysis based on the internal transcribed spacer (ITS) and 18S coding regions shows that Brachiaria endophyte isolates are genetically diverse, representing at least 10 distinct taxonomic groups (FIG. 1). B. humidicola and B. ruziziensis species, shown to produce brachialactone, exhibit high levels of fungal endophyte diversity and richness (FIG. 1).

Sequence data was used in BLASTN analysis to identify matches in the NCBI database. Brachiaria endophytes discovered are genetically novel. Comparison of each isolates ITS sequence to those in publically available databases did not identify any fungal strains with >90% identity. Phylogenetic analysis confirmed that isolates from different ITS clusters belonged to diverse genera. In several accessions, multiple endophytes isolated from a single plant belonged to different rDNA specific clusters, suggesting co-existence of multiple fungal endophyte species in the same plant (Table 6).

TABLE 6 Summary of fungal endophytes isolated from 11 Brachiaria-Urochola species. Host species Host plant identification No. of endophyte isolates B. decumbens 1.1 1 U. mosambicensis 2.1 23 B. holosericea 2.3 1 B. reptans 2.4 1 B. reptans 2.4 3 B. miliiformis 2.5 1 B. ruziziensis 2.6 2 B. ruziziensis 2.7 7 U. panicoides 2.8 3 U. mosambicensis 2.9 10 U. oligotricha 2.11 3 B. humidicola 2.12 3 U. panicoides 2.14 3 U. oligotricha 2.15 1 U. mosambicensis 3.3 3 B. humidicola 4.9 2 B. brizantha 5.1 4 B. decumbens 7.1 1 B. humidicola 8.1 3 B. humidicola 9.2 3 B. decumbens 10.1 1 U. mosambicensis 11.1 1 U. mosambicensis 12.1 5 B. decumbens 14.1 4 B. humidicola 15.2 3 B. distachya 8 2 B. miliiformis 26 3 Total Isolates 97

The rDNA-ITS region sequence for selected isolated and culturable fungal endophyte strains was used to identify their presence/absence in the microbiomes of 5 Brachiaria-Urochloa species (Bb—B. brizantha; Bh—B. humidicola; Bd—B. decumbens; Um—U. mosambicensis; Ur—U. ruziensis) (FIGS. 2-2A). A total of 27 isolates had sequence homology (>10e-145) to OTUs in the metagenomics analysis, further validating their endophytic ecological niche. The isolates had sequence homology to Acremonium sp., Sarocladium strictum, Hypocrea sp., Microsphaeropsis arundinis, an uncultured fungal sp. and Pseudogymnoascus sp. The pattern observed for the presence of ITS across host species follows within ITS Group similarity. For example, ITS5 (for example 2.15.A.2) and ITS1 form single ITS groups (no within group sub-clustering) and appear to be present ubiquitously in Brachiaria-Urochloa. In contrast, ITS7 shows patterns of host presence/absence that is related to the three ITS7 sub-clusters identified [represented by endophyte isolates 2.3.C.1 (cluster 1), 2.10.C.2 (cluster 2), 2.12.B.1 and 2.11.B.1 (cluster 3)]. The ITS6 group is genetically diverse, with three distinct clusters. Endophyte isolate 2.2.A.1 was detected in only 1 of 5 host species tested, while 2.10.D.1 shows a broad host range. The ITS2 Group endophytes exhibit two different host profiles. Endophytes 12.1.B and 9.2.B show a broad host range, being present in each of the 5 host species tested. In contrast 1.1.A shows a narrow host range, as it was detected in only 1 of 5 host species tested. Variation in host colonisation between putative sub-groups of the same taxonomic group may be an indicator of host-endophyte co-evolution and specialisation.

Example 3—Brachialactone is Microbial in Origin

Mature plants of Brachiaria-Urochloa grass-endophyte associations that had been maintained in a controlled environment were subjected to metabolic profiling analysis. Four individual plants (biological replicates) from each of three Brachiaria-Urochloa species (B. humidicola, U. mosambicensis, B. ruziziensis) were analysed for the presence of brachialactone using liquid chromatography-mass spectrometry (LC-MS). Freeze-dried pseudostem samples were prepared for LC-MS analysis using an 80% methanol extraction procedure. The compound brachialactone was identified in the root tissues of Brachiaria-endophyte associations (B. humidicola, B. ruziensis) (FIGS. 3 to 5; Table 7). The presence of brachialactone was confirmed through MS (ions extracted at the mass to charge ratio [m/z] of 333.2059 at RT 8.93 minutes). Brachialactone was not detected in B. decumbens-endophyte associations or U. mosambiciensis-endophyte associations (Table 7).

TABLE 7 Brachialactone detection in Brachiaria-Urochloa. Samples of B. humidicola-endophyte and B. ruzizensis-endophyte associations show presence of brachialactone. Brachiaria-Urochloa Brachialactone B. humidicola + U. mosambiciensis − B. decumbens − B. ruziziensis + +: brachialactone detected; −: no brachialactone detected

Example 4—an Optimised Method for Inoculation of Brachiaria Endophytes into Brachiaria-Urochloa

A host panel comprising commercially relevant Brachiaria-Urochloa germplasm was established to enable inoculation of genetically novel and highly diverse endophyte isolates into a single host genotype. An optimised method for endophyte inoculation into host plants free of microbial organisms in axenic conditions was developed, facilitating a high frequency of successful inoculation (Table 8). Four fungal endophytes representing four of the rDNA sequence-defined clades were identified as candidates for inoculation and characterisation into the Brachiaria-Urochloa host panel.

Sterilised Brachiaria seed are germinated under aseptic conditions to remove microbial organisms from the host plants to be used for inoculation. Microbe-free donor plantlets are grown on shoot multiplication media (M3B) under sterile conditions. Donor shoots are split into single tillers and transferred to root multiplication media (MS+NAA). Single tillers are grown for 2-3 weeks to promote root growth, plantlets are then again split into single tillers and the outer sheath is removed to reveal shoot initial. Shoot initials with intact roots are transferred to water agar for inoculation of endophyte mycelia. For endophyte inoculation, a small cut is made across the shoot meristem, and endophyte is inoculated into the wound. Following inoculation, plantlets are retained on ½ MS media for 2 weeks. They are then transferred to soil and grown under glasshouse conditions for 8 weeks before testing for endophyte presence using a diagnostic set of strain specific SSR markers (FIG. 6).

Endophyte inoculation frequency was determined for each candidate endophyte, approximately 6 months post inoculation, using a diagnostic (i.e. specific allele sizes at each SSR loci for each endophyte) set of simple sequence repeat (SSR) markers for each endophyte isolate.

Successful inoculation was achieved for representative endophytes from each of the ribosomal DNA sequence-defined clades (Table 8). Variation between endophyte isolates representing different ITS groups was observed. ITS5 (2.15.A.2)>ITS7 (2.10.C.2)>ITS2 (12.1.B)>ITS1 (5.1.B). Cross species compatibility was also observed. Endophyte isolate 2.15.A.2 (58 to 83%) and 2.10.C.2 (38% to 83%) exhibit broad species compatibility compared to the moderately compatible 12.1.B (8 to 76%) and narrow host compatibility of 5.1.B (0 to 7%). As would be expected, each endophyte strain shows highest inoculation frequency for the species from which it was originally isolated.

Variation in inoculation ability of the host was also observed. U. mosambicensis forms stable associations with a broad range of fungal endophytes at a very high frequency of successful inoculation (60%). Also of note is that U. mosambicensis forms associations with multiple, highly diverse fungal endophytes (FIG. 1; Table 6). Endophyte strains representing 6 of the 7 ITS groups identified in Brachiaria were isolated from this species (FIG. 1). B. humidicola forms stable associations with a broad range of fungal endophytes at a high frequency of successful inoculation (41%). As for U. mosambicensis, this species naturally harbours a diversity of multiple endophytes, with 4 of 7 ITS groups identified (FIG. 1).

Example 5—Metagenomics Analysis of the Brachiaria-Urochloa Seed Microbiome

A significant challenge in plant microbiome studies is that in order to analyse the endophytic component of a plant microbiome, it is necessary to extract DNA from plant tissues. The presence of a high proportion of plant DNA:microbe DNA (in the order of 20:1 for bacteria and 1:1 for fungi) in DNA extracted affects downstream sequence analysis. In previous studies, one way this has been dealt with is to generate large numbers of sequence reads to achieve a target number of microbiome reads.

In this example, a method was developed to enrich for the microbiome (both bacterial and fungal DNA) when extracting DNA from plant seed. The method is not limited in application to plant seed, and may be applied to any plant tissue from any species of interest, including leaf, stem and/or root plant material.

Seed-associated endophytic microbes are of interest as they may be exploited in a molecular breeding scenario whereby the microbe and host plant are co-selected for a particular trait of interest. Further, the presence of endophytic microbes in both root and seed microbiomes is of particular interest as seed associated microbes that are distributed throughout the plant may be associated with enhanced performance traits, such as pest and disease resistance, or biological nitrification inhibition (BNI) through the production of brachialactone.

Variation in host seed colonisation may be an indicator of host-endophyte co-evolution and specialisation

Once isolated and purified, individual components of the endophytic seed microbiome can be genome sequenced and characterised for molecular marker development, taxonomic identification and phylogenomic analyses. Selected microbiome component organisms can also be phenotypically assessed singly and in combination to identify microbes that confer enhanced production traits, for example BNI, to a range of commercially significant Brachiaria species. There is potential to further exploit the biological properties of the Brachiaria microbiome across a broad range of crop species to the benefit of sustainable agriculture and the environment.

Example 6—Methodology for Enriching for Bacterial and Fungal DNA in Microbiome Analysis of Seed

A method was developed to enrich for the microbiome (bacterial and fungal DNA) component in Brachiaria seed (FIG. 7). Although used for plant seed in this example, the method may also be applied to any plant tissue from any species of interest. Depending on the plant tissue, the person skilled in the art would understand that small changes may be made to the method to optimise the enrichment of the microbiome, for example the amount of grinding of the plant material may be varied e.g. from finely ground to roughly ground, depending on the nature of the plant material.

Fifty grams of seed from each of the ten selected accessions (Table 9) was surface sterilised (5% [w/v] NaHCl and Tween 20) for 30 min with shaking. Seed samples were then rinsed eight to ten times in sterile MilliQ water to ensure the sterilant had been completely removed. Seed were dried on sterile filter paper under aseptic conditions overnight. Dried seeds were partially ground using a genogrinder (SPEX SamplePrep 2010 Geno/Grinder®, Metuchen, USA). Ground seeds were then washed twice for 12 hrs with absolute ethanol and continuous shaking. Following washes, samples were allowed to settle and the supernatant containing the seed associated endophytic microbiome collected. The supernatant was then completely evaporated under sterile conditions. DNA was extracted from the final crude (what was left following ethanol evaporation) using the Qiagen DNeasy plant mini kit according to manufacturer's instructions.

Simultaneously, DNA was also extracted from surface sterilised seeds (10 seeds from each accession) using the Qiagen DNeasy plant mini kit according to manufacturer's instructions. Bacteria and fungi were evaluated in the metagenomics analyses using the universal PCR primers 515f (Wang & Qian, 2009) and 806r (McBain et al, 2003) for profiling the bacterial microbiome (V4 region of the 16S rDNA gene, approx. 350 base pairs), and 58A2F (Martin & Rygiewicz, 2005) and ITS4 (White et al, 1990) for the fungal microbiome (ITS2 region of the rDNA genes, approx. 400 base pairs), with associated Illumina adapters. Ribosomal RNA gene amplicons were prepared and sequenced on the MiSeq (Illumina) according to the corresponding user guide.

TABLE 9 Brachiaria species used in this example. Records for Accessions used in this study BNI compound* Each accession refers to a Brachiaria species production different seed batch B. brizantha No 5 59591 B. humidicola Yes 2 4 8 9 15 B. decumbens Yes 7 13 B. ruziziensis Yes 30623 *BNI compound: Biological nitrification inhibition compounds e.g. brachialactone

Example 7—Metagenomics Analysis to Identify the Seed Associated Endophytic Microbiome of Brachiaria Grasses

The endophytic (bacterial and fungal) seed microbiomes of four selected Brachiaria-Urochloa species were profiled using metagenomics with the aim of identifying microbes associated with a particular species and or/trait, such as pest and disease resistance, or biological nitrification inhibition (BNI) through the production of brachialactone.

Brachiaria species examined included three species—B. humidicola, B. ruziziensis, B. decumbens—previously documented to produce biological nitrification inhibition (BNI) compounds, and B. brizantha which does not produce BNI compounds (Table 9).

The data is then analysed to identify the seed associated endophytic microbiome of Brachiaria:

-   -   associated with the production of brachialactone         -   specifically with the BNI trait in B. humidicola         -   in general with the BNI trait (i.e. common to B.             humidicola, B. ruziziensis and B. decumbens)     -   unique to B. humidicola, B. ruziziensis, B. decumbens or B.         brizantha     -   in common to all Brachiaria species studied

It is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to be in any way limiting or to exclude further additives, components, integers or steps.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and/or regarded as relevant by a person skilled in the art.

REFERENCES

-   1. de Boer, A. H., & de Vries-van Leeuwen, I. J. “Fusicoccanes:     diterpenes with surprising biological functions”, Trends in Plant     Science, 2012, 17(6), 360-368. -   2. Subbarao, D. V., et al. “A bioluminescence assay to detect     nitrification inhibitors released from plant roots: a case study     with Brachiaria humidicola”, Plant Soil, 2006, 288, 101-112. -   3. Subbarao, G. V., et al. “Evidence for biological nitrification     inhibition in Brachiaria pastures”, Proceedings of the National     Academy of Sciences of the United States of America, 2009, 106(41),     17302-17307. -   4. White, T. J. et al. “Amplification and direct sequencing of     fungal ribosomal RNA genes for phylogenetics”, In PCR Protocols: A     Guide to Methods and Applications, 1990, pp. 315-322, Academic     Press. -   5. Martin K J, Rygiewicz P T (2005) Fungal-specific PCR primers     developed for analysis of the ITS region of environmental DNA     extracts. BMC Microbiology 5: 28. -   6. McBain A J, Bartolo R G, Catrenich C E, Charbonneau D, Ledder R     G, Rickard A H, Symmons S A, Gilbert P (2003) Microbial     characterization of biofilms in domestic drains and the     establishment of stable biofilm microcosms. Applied and     Environmental Microbiology 69: 177-185. -   7. Wang Y, Qian P-Y (2009) Conservative fragments in bacterial 16S     rRNA genes and primer design for 16S ribosomal DNA amplicons in     metagenomic studies. PloS one 4: e7401. 

1-13. (canceled)
 14. A substantially purified or isolated endophyte selected from the group consisting of Hypocrea sp./Acremonium sp. 2.15.A.2, Acremonium sp. 2.3.C.1, Microsphaeropsis arundis 2.10.D.1, Sarocladium sp./Acremonium sp. 2.12.B.1, Sarocladium sp./Acremonium sp. 2.10.C.2 and Sarocladium sp./Acremonium sp. 2.11.B.1, as deposited at the National Measurement Institute with accession numbers V15/028237, V15/028238, V15/028239, V15/028240, V15/028241 and V15/028242, respectively.
 15. A plant inoculated with one or more endophytes according to claim 14, said plant comprising an endophyte-free host plant stably infected with said endophyte.
 16. An inoculated plant according to claim 15, which has beneficial properties when compared with an uninoculated control plant, said beneficial properties being improved tolerance to water and/or nutrient stress or improved resistance to pests and/or diseases.
 17. An inoculated plant according to claim 15, which has beneficial properties when compared with an uninoculated control plant, said beneficial properties being the production of a fusicoccane compound.
 18. An inoculated plant according to claim 17, wherein the fusicoccane compound is brachialactone.
 19. An inoculated plant according to claim 15, wherein the plant is a grass species plant.
 20. An inoculated plant according to claim 19, wherein the grass species plant is selected from the group consisting of Brachiaria brizantha, Brachiaria decumbens, Brachiaria humidicola, Brachiaria stolonifera, Brachiaria ruziziensis, B. dictyoneura, Urochloa brizantha, Urochloa decumbens, Urochloa humidicola, Urochloa mosambicensis, interspecific and intraspecific hybrids of Brachiaria-Urochloa species complex, and those belonging to the genera Lolium, Festuca, Triticum, Hordeum, Sorghum, Zea, Oryza, Saccharum, Paspalum, Miscanthus, Panicum, Pennisetum, Poa, Eragrostis and Agrostis.
 21. A plant infected with an endophyte according to claim 14, wherein the plant is infected by a method selected from the group consisting of: inoculation, breeding, crossing, hybridisation, transduction, transfection, transformation and/or gene targeting; and combinations thereof. 22-44. (canceled) 